Defining characteristics of the mammalian nervous system include the vast diversity of its neuronal cell types and the exquisite specificity with which they interact to form trillions of synapses during development. It is widely believed that molecular diversity, provided by combinatorial interactions between multiple receptors and cell adhesion molecules (CAMs), is required for the specificity of cell recognition. Our prior work has identified the 22 gamma protocadherins (gamma -Pcdhs), encoded by the Pcdhg gene cluster, as top candidates for providing such molecular diversity because they mediate homophilic trans-interactions as combinatorially diverse (104-105) cis-tetramers. While studies on existing Pcdhg null mice demonstrate that these molecules are critical for neurodevelopment, the extent to which gamma -Pcdh isoform diversity plays a role in their functions remains a critical unknown that has not been addressed due to constraints of standard knockout mouse generation. The long- term goal of our research is to elucidate mechanisms of molecular diversity in regulating critical aspects of neurodevelopment relevant to the etiology of human disorders such as autism and intellectual disability. The objective of this application is to utilize CRISPR/Cas9 technology to test the hypothesis that isoform diversity of the gamma -Pcdhs is required for their many functions in vivo. We will pursue this objective through two Specific Aims: 1) Use the CRISPR/Cas9 system to reduce the molecular diversity of functional Pcdhg isoforms in the mouse genome; 2) Determine the extent to which the isoform diversity of gamma -Pcdhs is required for their known roles in neurodevelopment. In studies of the Drosophila Dscam1 gene, which can generate 19,008 distinct adhesive isoforms through combinatorial alternative splicing, a key breakthrough was the generation of mutant strains with reduced diversity of alternate exons. An analogous approach is needed for the Pcdhg locus in mice, but requires new, higher-throughput techniques for the generation of mutant lines. The recently- described CRISPR-Cas9 system holds great promise for the simultaneous targeting of multiple sites in the genome for the generation of indel mutations that will disrupt genes. We will design CRISPR guide RNA sequences targeting each of the 22 Pcdhg variable exons, and will inject them into fertilized mouse oocytes to generate a library of mouse lines in which varying numbers of these exons are disrupted. A subset of these lines will be strategically chosen (covering a wide range of intact Pcdhg exon diversity) and known in vivo phenotypes in neuronal survival, dendrite and axon arborization, and synaptogenesis will be analyzed quantitatively. In this way, we can define the extent to which each neuronal phenotype depends on gamma -Pcdh isoform diversity, and better understand the mechanisms through which these critical CAMs act. The proposed studies will further provide an important in vivo test of the specificity and efficiency o the CRISPR/Cas9 system for simultaneous gene targeting, of importance for the advancement of reverse genetics in the mouse.